A Pseudovirus-Based Entry Assay to Evaluate Neutralizing Activity against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infection can cause life-threatening pneumonia and bronchiolitis, posing a significant threat to human health worldwide, especially to children and the elderly. Currently, there is no specific treatment for RSV infection. The most effective measures for preventing RSV infection are vaccines and prophylactic medications. However, not all population groups are eligible for the approved vaccines or antibody-based preventive medications. Therefore, there is an urgent need to develop novel vaccines and prophylactic drugs available for people of all ages. High-throughput assays that evaluate the efficacy of viral entry inhibitors or vaccine-induced neutralizing antibodies in blocking RSV entry are crucial for evaluating vaccine and prophylactic drug candidates. We developed an efficient entry assay using a lentiviral pseudovirus carrying the fusion (F) protein of type A or B RSV. In addition, the essential parameters were systematically optimized, including the number of transfected plasmids, storage conditions of the pseudovirus, cell types, cell numbers, virus inoculum, and time point of detection. Furthermore, the convalescent sera exhibited comparable inhibitory activity in this assay as in the authentic RSV virus neutralization assay. We established a robust pseudovirus-based entry assay for RSV, which holds excellent promise for studying entry mechanisms, evaluating viral entry inhibitors, and assessing vaccine-elicited neutralizing antibodies against RSV.

A new intracellular targeting motif in the cytoplasmic tail of the spike protein may act as a target to inhibit SARS-CoV-2 assembly.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a threat to global public health, underscoring the urgent need for the development of preventive and therapeutic measures. The spike (S) protein of SARS-CoV-2, which mediates receptor binding and subsequent membrane fusion to promote viral entry, is a major target for current drug development and vaccine design. The S protein comprises a large N-terminal extracellular domain, a transmembrane domain, and a short cytoplasmic tail (CT) at the C-terminus. CT truncation of the S protein has been previously reported to promote the infectivity of SARS-CoV and SARS-CoV-2 pseudoviruses. However, the underlying molecular mechanism has not been precisely elucidated. In addition, the CT of various viral membrane glycoproteins play an essential role in the assembly of virions, yet the role of the S protein CT in SARS-CoV-2 infection remains unclear. In this study, through constructing a series of mutations of the CT of the S protein and analyzing their impact on the packaging of the SARS-CoV-2 pseudovirus and live SARS-CoV-2 virus, we identified V1264L1265 as a new intracellular targeting motif in the CT of the S protein, that regulates the transport and subcellular localization of the spike protein through the interactions with cytoskeleton and vesicular transport-related proteins, ARPC3, SCAMP3, and TUBB8, thereby modulating SARS-CoV-2 pseudovirus and live SARS-CoV-2 virion assembly. Either disrupting the V1264L1265 motif or reducing the expression of ARPC3, SCAMP3, and TUBB8 significantly repressed the assembly of the live SARS-CoV-2 virion, raising the possibility that the V1264L1265 motif and the host responsive pathways involved could be new drug targets for the treatment of SARS-CoV-2 infection. Our results extend the understanding of the role played by the S protein CT in the assembly of pseudoviruses and live SARS-CoV-2 virions, which will facilitate the application of pseudoviruses to the study of SARS-CoV-2 and provide potential strategies for the treatment of SARS-CoV-2 infection.

Epidemiological and Whole-Genome Sequencing Analysis of a Gastroenteritis Outbreak Caused by a New Emerging Serotype of Vibrio parahaemolyticus in China.

Vibrio parahaemolyticus is an important foodborne pathogen with diverse serotypes. In May 2021, we investigated a gastroenteritis outbreak that occurred in China, caused by V. parahaemolyticus O10:K4 infection. Based on the epidemiological curve, this outbreak was identified as a homologous exposure event. A case-control study demonstrated that emperor crab with mashed garlic (odds ratio [OR] = 4.60, p = 0.030; 95% confidence interval [95% CI]: 1.11-19.14), goose liver geoduck (OR = 4.50, p = 0.029; 95% CI: 1.12-18.13), shrimp (OR = 4.89, p = 0.021; 95% CI: 1.22-19.65), and sea cucumber (OR = 7.36, p = 0.005; 95% CI: 1.68-32.26) were the potential sources of the food poisoning. V. parahaemolyticus isolates from 18 laboratory-confirmed cases were all serotyped O10:K4, and determined to be sequence type ST3 via multilocus sequence typing. Pulsed field gel electrophoresis and whole-genome sequencing analysis revealed the identical pattern and 0-2 single nucleotide variation among these isolates. tdh was positive in all isolates, while trh and Orf8 were absent. Seven essential base positions in toxRS for pandemic clone identification were identical between the O10:K4 and O3:K6 pandemic clones. Phylogenetic analysis with 45 additional genomes of 13 different serotypes showed the closest genetic relationship between O10:K4 and O1: KUT. O10:K4 was thought to evolve from the O3:K6 pandemic clone. The new serovariant of O3:K6 poses a challenge for the prevention and control of V. parahaemolyticus disease outbreaks, or even epidemics, in the future.

The E484K Substitution in a SARS-CoV-2 Spike Protein Subunit Vaccine Resulted in Limited Cross-Reactive Neutralizing Antibody Responses in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially emerging variants, poses an increased threat to global public health. The significant reduction in neutralization activity against the variants such as B.1.351 in the serum of convalescent patients and vaccinated people calls for the design of new potent vaccines targeting the emerging variant. However, since most vaccines approved and in clinical trials are based on the sequence of the original SARS-CoV-2 strain, the immunogenicity and protective efficacy of vaccines based on the B.1.351 variant remain largely unknown. In this study, we evaluated the immunogenicity, induced neutralization activity, and protective efficacy of wild-type spike protein nanoparticle (S-2P) and mutant spike protein nanoparticle (S-4M-2P) carrying characteristic mutations of B.1.351 variant in mice. Although there was no significant difference in the induction of spike-specific IgG responses in S-2P- and S-4M-2P-immunized mice, neutralizing antibodies elicited by S-4M-2P exhibited noteworthy, narrower breadth of reactivity with SARS-CoV-2 variants compared with neutralizing antibodies elicited by S-2P. Furthermore, the decrease of induced neutralizing antibody breadth at least partly resulted from the amino acid substitution at position 484. Moreover, S-4M-2P vaccination conferred insufficient protection against live SARS-CoV-2 virus infection, while S-2P vaccination gave definite protection against SARS-CoV-2 challenge in mice. Together, our study provides direct evidence that the E484K substitution in a SARS-CoV-2 subunit protein vaccine limited the cross-reactive neutralizing antibody breadth in mice and, more importantly, draws attention to the unfavorable impact of this mutation in spike protein of SARS-CoV-2 variants on the induction of potent neutralizing antibody responses.

Salmonella enterica subsp. diarizonae Harboring ST233, ST1263, and ST1845 in Children.

Objective: This study aims to analyze the molecular epidemiology, resistance, and pathogenicity of Salmonella enterica subsp. diarizonae isolated from children. Methods: Whole genome sequencing was carried out, and molecular serotypes, sequence types, resistance genes, and virulence genes of S. enterica subsp. diarizonae isolates were analyzed. Antimicrobial susceptibility test was determined by commercialized microdilution method. Results: A total of three isolates of S. enterica subsp. diarizonae were isolated during 2015 to 2020. The molecular serotypes of the three strains were 61:c:z35, 61:l,v:1,5,7:[z57], and 65:k:z, respectively, and the sequence types were ST1845, ST233, and ST1263. All the three isolates were susceptible to ceftriaxone, ceftazidime, cefepime, amoxycillin/clavulanic acid, piperacillin/tazobactam, ertapenem, imipenem, levofloxacin, and trimethoprim/sulfamethoxazole. No other resistant gene was detected except aac(6')-Iaa. There were no resistant plasmids detected in all the three isolates. A total of 76 genes were present in all isolates, containing 49 genes of Type III Secretion System (T3SS) mediated by SPI-1and SPI-2, 13 genes of adherence (type 1 fimbriae, Agf, and MisL-related genes), 11 genes of iron uptake (Yersiniabactin), two genes of magnesium uptake, and one gene of typhoid toxin(cdtB). Conclusion: The serotypes and sequence types of S. enterica subsp. diarizonae isolates were rarely reported in children; all the S. enterica subsp. diarizonae isolates were susceptible to detected antibiotics; T3SS, adherence, iron uptake, magnesium uptake, and typhoid toxin were responsible for pathogenicity of the S. enterica subsp. diarizonae isolates in children.

Comparative genomic analyses of IncpA1763-KPC plasmids.

Multi-replicon plasmids harboring the IncpA1763-KPC replicon together with other replicons are being increasingly reported among Enterobacteriaceae species. However, plasmids with single IncpA1763-KPC replicons are poorly studied as a different incompatibility (Inc) group, despite their rise in appearance in some strains. IncpA1763-KPC plasmids, pA1763-KPC, and p427113-2, from two clinical Klebsiella pneumoniae isolates were fully sequenced by high-throughput genome sequencing. Linear structural comparisons of IncpA1763-KPC backbone region were made between these two plasmids and six arbitrarily selected representative IncpA1763-KPC plasmids sequenced previously. A further detailed genomic comparison was carried out between plasmids pA1763-KPC, p427113-2, and pFB2.2, which show high homology across the backbone sequence to one another. Among all sequenced IncpA1763-KPC plasmids considered in this study, plasmids pA1763-KPC and p427113-2 showed the most complete IncpA1763-KPC backbones. These were composed of the IncpA1763-KPC replicon (repAIncpA1763-KPC and its iterons), the 5.6-kb IncpA1763-KPC -type maintenance region, the 27.7-kb IncFIIK -type maintenance region, and the 36.6-kb IncFIIK -type conjugal transfer regions. Compared with pA1763-KPC or p427113-2, the backbone regions of the other analyzed IncpA1763-KPC plasmids had gradually undergone different deletions or truncations, but shared small and core IncpA1763-KPC backbones including the IncpA1763-KPC replicon, IncpA1763-KPC -type maintenance region, and residual IncFIIK -type maintenance region. Accessory modules integrated into IncpA1763-KPC backbones included the multidrug-resistant module blaKPC-2 region in pA1763-KPC, the metal-resistance modules ars region together with ncr region in pFB2.2 and sil in pKPN-9a0d, the ISKpn14-to-IS26 region in p427113-2, and other non-resistance region in the respective plasmids. This detailed comparative genomics analysis of IncpA1763-KPC plasmids provides a deep insight into their diversification and evolution.

Prevalence and Population Analysis of Vibrio parahaemolyticus Isolated from Freshwater Fish in Zhejiang Province, China.

Objectives: The previous researches revealed that Vibrio parahaemolyticus has been detected in freshwater fish samples. However, the molecular characteristics of V. parahaemolyticus isolated from freshwater fish, including pathogenic and pandemic strains, are still unknown. This study aims to characterize and identify molecular properties of the bacterium. In addition, it identifies the source of V. parahaemolyticus from freshwater fish samples in Zhejiang Province, China. Methods: Four hundred and twenty-one freshwater fish samples (from fishing farms, retail markets, and restaurants) and 212 seafood samples (from retail markets) were collected in 10 cities of Zhejiang Province. V. parahaemolyticus strains were isolated from these samples and comparatively analyzed by multilocus sequence typing, serotyping, antimicrobial susceptibility test, and polymerase chain reaction, targeting common toxin genes (tdh, trh) and markers for pandemic strains (orf8, toxRS/new). Results: Sixty-eight V. parahaemolyticus strains were isolated from the 421 freshwater fish samples, and 89 V. parahaemolyticus isolates were identified out of 212 seafood samples. The detection rate of V. parahaemolyticus was significantly different (p < 0.05) between the fishing farms, the retail markets, and the restaurants. The isolates from freshwater fish samples were divided into eight O serotypes with three O3:K6 isolates, which contain three pandemic complexes (tdh+, orf8+, toxRS/new+). A total of 53 different sequence types (STs) were identified among the 68 isolates, including 28 novel STs. Antimicrobial susceptibility results indicated that 76.5% of the strains were resistant to ampicillin. A third (3/9) of the isolates from fishing farm sources shared the same STs with their counterparts from retail markets. Compared with the isolates from the seafood samples collected in the same sampling sites, 13.2% (9/68) freshwater fish isolates overlapped with seafood isolates. Conclusions: Our study showed that V. parahaemolyticus population in freshwater fish is genetically diverse. The V. parahaemolyticus contaminates might have come from both fishing farm sources and cross-contamination from seafood in the closed area at the markets. Freshwater fish may work as a reservoir of pathogenic and pandemic V. parahaemolyticus isolates, indicating potential public health and food safety risks associated with the consumption of freshwater fish.

The Epidemiology of Meningococcal Disease and Carriage, Genotypic Characteristics and Antibiotic Resistance of Neisseria meningitidis Isolates in Zhejiang Province, China, 2011-2021.

Neisseria meningitidis (Nm) remains a worldwide leading cause of epidemic meningitis. During 2011-July 2021, 55 meningococcal disease (MD) cases were reported with a case fatality rate of 5.45% in Zhejiang Province, China. The median age was 7 years. The annual incidence was 0.0017-0.0183 per 100,000 population. The highest age-specific incidence was observed in the group younger than 1 year. Serogroup was identified in 30 laboratory-confirmed MD cases, and MenB was most predominant. MenB was mainly observed in two age groups: younger than 5 and older than 35 years. MenB incidence was significantly increasing from 0.0018 per 100,000 in 2013 to 0.0070 per 100,000 in 2019. During 2015-2020, 17 positive samples were detected from 2,827 throat swabs from healthy population, of which 70.59% was MenB. Twenty multilocus sequence typing sequence types (STs) containing eight newly assigned STs (ST15881-ST15888) were determined in all Nm isolates. Either in MD cases or in healthy population, MenB CC ST-4821 was the predominant ST. It was worth noting that two MenY CC ST-23 cases occurred in 2019 and 2021, respectively. MenY CC ST-23 MD cases increased gradually in China. Phylogeny results based on genome sequencing indicated that Chinese MenW CC ST-11 isolates were genetically linked and grouped together with Japanese isolates, separated from MenW CC ST-11 isolates from Saudi Arabia Hajj outbreak, Europe, South Africa, South America, North America, and Oceania. MenW CC ST-11 isolates from East Asia might have evolved locally. Antibiotic susceptibility tests revealed a relatively high resistance rate (22.86%) of Nm isolates to penicillin. This study provided valuable data for Chinese public health authorities to grasp the temporal epidemiological characteristics of MD and healthy carriage.

Molecular Characterization and Antibiotic Resistant Profiles of Campylobacter Species Isolated From Poultry and Diarrheal Patients in Southeastern China 2017-2019.

Campylobacter is a zoonotic pathogen that causes foodborne diarrheal illness globally. To better understand health risks in Southeastern China, Campylobacter spp. were surveyed in humans and representative poultry products over 3 years. One hundred and ninety-five representative isolates (n = 148, Campylobacter jejuni; n = 45, Campylobacter coli; n = 2 Campylobacter hyointestinalis) were examined for genetic relatedness and antimicrobial susceptibility. Nearly all Campylobacter isolates (99.0%, 193/195) were resistant to at least one class of antimicrobials, and 45.6% (89/195) of the isolates exhibited multidrug resistance. Genotypic analysis revealed high diversity among tested strains. Multilocus sequence typing (MLST) displayed 120 sequence types (STs) including 42 novel STs being added to the PubMLST international database. Sixty-two STs belonged to 16 previously characterized clonal complexes (CCs), of which CC-21, CC-45, CC-464, CC-574, CC-353, and CC-828 were most frequently identified. In addition, pulsed-field gel electrophoresis (PFGE) fingerprinting resulted in 66 PFGE SmaI patterns among the 125 isolates, with eight patterns shared between human and poultry sources. Subtyping data did not correlate with antimicrobial resistance phenotypes. Taken together, this large-scale surveillance study highlights high antimicrobial resistance and molecular features of Campylobacter isolates in Southeastern China.

Epidemiological and Whole Genomic Sequencing Analysis of a Campylobacter jejuni Outbreak in Zhejiang Province, China, May 2019.

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide with a very low outbreak reported in China. In May 2019, we investigated an outbreak of Campylobacter jejuni infections among students in a junior high school in Eastern China. Cases were interviewed to identify a common source of contamination. As cases were identified in the same school during a period of time, menus were reviewed and food items included in the questionnaire. Rectal swabs from school kitchen staff and suspected food items (raw chicken) from a local market from where the school food came were examined for C. jejuni. Pulsed-field gel electrophoresis and whole genome sequencing were performed to determine the relatedness of the isolates. To identify the source of the contamination, a case-control study was conducted. Forty-five cases were reported with diarrhea among 1696 students and staff. Stool samples for 10 of the 45 and 5 tested positive for C. jejuni. WGS analysis revealed a 0-4 single nucleotide variation in case-patient isolates. Although we were unable to identify the specific food item, a specific menu was identified as the potential source of the contamination (odds ratios = 20.82; 95% confidence interval = 6.472-66.957). In this menu, chicken was served. A food isolate collected from chicken in Zhejiang province in 2018 was positive for the same identical strain (5-7 single nucleotide polymorphisms). This is one of the few reports in China about outbreak caused by C. jejuni. This investigation illustrates the potential risk of outbreaks caused by Chinese cold dishes of chicken.

Prevalence and genetic characteristics of Cronobacter spp. from food and human clinical stool samples in Wenzhou, China 2008-2018.

Pathogenic Cronobacter species are responsible for life-threatening illness in neonates. A ten-year comprehensive survey was conducted to examine the population structure and antimicrobial resistant patterns of Cronobacter isolates from food (n = 78) and clinical (n = 12) sources in Wenzhou, China. A total of 90 (4.4%) isolates were recovered from 2051 collected samples. The occurrence of Cronobacter spp. was highest in spices with a rate of 22% (26/119), whereas the lowest contamination rate of 1% was found in powered infant and toddler formula (7/494), special medical infant formula (1/95) and human stool samples (12/1024). Cronobacter strains revealed a high degree of genetic diversity among the isolates tested. Pulsed-field gel electrophoresis (PFGE) distinguished 75 clonal groups, and the biggest cluster consisted of four strains. Multilocus sequence typing (MLST) method displayed 43 sequence types (STs), of which ST1, ST4, ST8, ST64, ST148 and ST201 were most frequently identified. Meanwhile, two new sequence types were discovered and added to the PubMLST international database. Resistance to ceftriaxone, cefotaxiv, amoxicillin, ampicillin, cefoxitin, tetracycline, streptomycin, azithromycin, chloramphenicol, as well as multidrug resistance, was noted. Taken together, this large-scale surveillance study highlights the wide dissemination and diverse molecular features of Cronobacter spp. in Wenzhou China.

Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning.

Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus.

Prevalence, Genotypic Characteristics and Antibiotic Resistance of Listeria monocytogenes From Retail Foods in Bulk in Zhejiang Province, China.

Listeria monocytogenes is an important foodborne pathogen causing public concern. A total of 3354 retail foods in bulk were sampled and screened for L. monocytogenes. Seventy-three (2.2%) samples including 21 ready-to-eat (RTE) foods and 52 raw foods were confirmed positive for L. monocytogenes. Sushi and salmon sashimi occupied the top two slots in RTE foods with relatively high presence rate of 12.9 and 6.9%, respectively. Meanwhile, L. monocytogenes was found to be distributed unequally in raw foods; the presence rates in raw meat (3.5%) and poultry (3.8%) were significantly higher than that in raw seafood (1.3%). Notably, L. monocytogenes was not detected in raw freshwater food. The L. monocytogenes isolates belonged to four serotypes, 1/2a, 1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). Eighteen sequence types (STs) and eighteen virulence types (VTs) containing four newly assigned VTs (VT180, VT181, VT182, and VT183) were determined via multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST). Among the 73 L. monocytogenes isolates, 23 (31.5%) belonged to epidemic clones (ECs) including ECI, ECIV, ECV, ECVI, ECVIII and ECXI among which ECV was predominant. Antibiotic susceptibility tests revealed a high resistance rate (11.0%) to tetracycline. Moreover, we identified the distribution patterns of virulence genes of four Listeria pathogenicity islands (LIPI) in L. monocytogenes isolates. prfA, hly, plcA, plcB, mpl, actA genes in LIPI-1 and inlA, inlB, inlC, inlJ genes in LIPI-2 were detected in approximately all L. monocytogenes isolates. The distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with lineage and ST. LIPI-4 genes were present exclusively in ST87 isolates. Relatedness analysis revealed the absence of distinct association between STs, ECs, LIPI-3 and LIPI-4 distribution and specific food groups. This study provided fundamental data for Chinese food safety authorities to grasp the contamination status of L. monocytogenes in foods, assess the potential risk of this pathogen and further address the safety issue of retail foods in bulk in China.

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems.

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes. In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

Capsule switching of Neisseria meningitidis sequence type 7 serogroup A to serogroup X.

OBJECTIVES: The bacterial pathogen Neisseria meningitidis is able to escape the currently available capsule-based vaccines by undergoing capsule switching. In this study, we investigated whether capsule switching has occurred in a recently emerged sequence type (ST) 7 serogroup X isolate in China, for which currently no vaccine is available. METHODS: To identify capsule switching breakpoints, the capsule locus and flanking regions of the ST-7 serogroup X isolate and three endemic ST-7 serogroup A isolates were sequenced and compared. To obtain further insight into capsule switching frequency and length of DNA fragments involved, capsule switching assays were performed using genomic DNA containing combinations of antibiotic selection markers at various locations in the capsule locus and flanking regions. RESULTS: Sequence analyses showed that capsule switching has occurred and involved a 8450 bp serogroup X DNA fragment spanning the region from galE to ctrC. Capsule switching assays indicate that capsule switching occurs at a frequency of 6.3 × 10-6 per bacterium per µg of DNA and predominantly involved DNA fragments of about 8.1-9.6 kb in length. CONCLUSIONS: Our results show that capsule switching in N. meningitidis occurs at high frequency and involves recombination in the flanking regions of the capsule biosynthesis genes.

Quantitative Prevalence, Phenotypic and Genotypic Characteristics of Bacillus cereus Isolated from Retail Infant Foods in China.

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 103-104 CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China.

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates.

Genotypic heterogeneity of emetic toxin producing Bacillus cereus isolates from China.

Emetic toxin-producing Bacillus cereus (emetic B. cereus) is the third member of B. cereus group whose toxins are encoded by megaplasmids, beside anthrax and insecticidal toxins of B. anthracis and B. thuringiensis, respectively. A total of 18 emetic isolates collected from food poisoning events, clinical and non-random food samples in Zhejiang province of China, were analyzed by plasmid screening, pulse field gel electrophoresis, multilocus sequence typing, and toxic gene identification to investigate their genotypic diversity. In this study, 13 plasmid profile types, 14 pulse types and 6 different STs from emetic isolates were detected, in which ST 1035,1038,1053,1054 and 1065 were first assigned and reported. The toxic gene ces existed on its own, or coexisted with other toxic genes bceT, cytk, entFM and nhe, but never with hbl in emetic isolates. The results demonstrated that the emetic B. cereus strains from China were heterologous at genotypic level.

Genome Sequence of Bacillus cereus Phage vB_BceS-MY192.

ITALIC! Bacillus cereusis an opportunistic foodborne pathogen. The phage vB_BceS-MY192 was isolated from ITALIC! B. cereus192 in a cooked rice sample. The temperate phage belongs to the ITALIC! Siphoviridaefamily, ITALIC! Caudoviralesorder. Here we announce the phage genome sequence and its annotation, which may expand the understanding of ITALIC! B. cereussiphophages.

Glioma Association and Balancing Selection of ZFPM2.

ZFPM2, encoding a zinc finger protein and abundantly expressed in the brain, uterus and smooth muscles, plays important roles in cardiac and gonadal development. Abnormal expression of ZFPM2 in ovarian tumors and neuroblastoma has been reported but hitherto its genetic association with cancer and effects on gliomas have not been studied. In the present study, the hexamer insertion-deletion polymorphism rs71305152, located within a large haplotype block spanning intron 1 to intron 3 of ZFPM2, was genotyped in Chinese cohorts of glioma (n = 350), non-glioma cancer (n = 354) and healthy control (n = 463) by direct sequencing and length polymorphism in gel electrophoresis, and ZFPM2 expression in glioma tissues (n = 69) of different grades was quantified by real-time RT-PCR. Moreover, potential natural selection pressure acting on the gene was investigated. Disease-association analysis showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.016), and the heterozygous genotype compared to the combined homozygous genotypes was less frequent in gliomas than in controls (P = 0.005) or non-glioma cancers (P = 0.020). ZFPM2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.002), with higher expression levels in the low-grade gliomas. In the astrocytoma subtype, higher ZFPM2 expression was also correlated with the rs71305152 heterozygous genotype (P = 0.028). In addition, summary statistics tests gave highly positive values, demonstrating that the gene is under the influence of balancing selection. These findings suggest that ZFPM2 is a glioma susceptibility gene, its genotype and expression showing associations with incidence and severity, respectively. Moreover, the balancing selection acting on ZFPM2 may be related to the important roles it has to play in multiple organ development or associated disease etiology.